The regulation of glucose homeostasis is a complex process, which is disrupted in disease states such as type 2 diabetes. Insulin is the primary hormone that regulates glucose homeostasis. Insulin stimulates glucose uptake in muscle and fat by causing the movement of intracellular membranes containing GLUT4 glucose transporters, which fuse and insert GLUT4 at the cell surface. This effect of insulin is impaired in the setting of overnutrition, inactivity, and genetic predisposition, resulting in insulin resistance and contributing to the development of diabetes. Therefore, to understand the pathogenesis of metabolic disease, it is necessary to understand the molecular mechanisms that specify the trafficking of GLUT4 among intracellular membranes, and by which this trafficking is modulated by insulin and disrupted in insulin resistance. Previous work by this project identified the TUG protein as a major regulator of GLUT4 trafficking and glucose uptake in muscle and fat cells. The data support a model in which TUG mediates the intracellular retention of GLUT4, together with selected other proteins, in specific membrane vesicles within unstimulated cells. Insulin then mobilizes these vesicles by triggering TUG endoproteolytic cleavage. Cleavage coordinates glucose uptake with other physiologic effects, resulting from the action of proteins that are co-regulated with GLUT4 by this mechanism, as well as with possible effects on energy expenditure. In insulin resistant individuals, alterations in this membrane trafficking mechanism may contribute to multiple aspects of the metabolic syndrome. Yet, it remains unknown where TUG and GLUT4 are localized in cells, how this localization is affected in insulin resistance, and whether attenuated TUG cleavage can cause insulin resistance in vivo. To address these questions, three Aims will be undertaken. Aim 1 will characterize how TUG is able to trap the GLUT4-containing vesicles in unstimulated cells, and how it maintains these vesicles in an insulin-responsive configuration. Aim 2 will study the location and mechanism by which TUG anchors these vesicles to intracellular structures, and how this may be affected in insulin resistance. . Aim 3 will study the importance of TUG proteolysis in muscle for overall insulin action and glucose homeostasis. We anticipate that, together, these studies will result in an improved understanding of molecular mechanisms regulating glucose metabolism and energy expenditure, with implications for the prediction, prevention, and treatment of diabetes and the metabolic syndrome.